Although no crystal structure of mammalian hexokinase is available, extensive work on the characterization of hexokinase has been carried out. Questions remain on the number and position of disulfide bonds present in this enzyme containing 27 cysteines. Our goal is to provide this information using MALDI-MS. Technology is now available to address structural questions on proteins of this size (more than 900 residues). The first step in this project is to perform standard proteolytic digestions of the enzyme and use MALDI to determine the masses of the digestion products that can be desorbed and ionized in the experiment. The complete digestion is first being analyzed as a single, complex mixture. Next, a simple HPLC fractionation will be performed to determine whether the full set of digestion products can be detected in a single experiment, or if some components suppress the detection of others. We will evaluate whether disulfide bond cleavage and scrambling occurs in the digestion or ionization process. Once the situation has been defined, experimental modifications can be designed to give the desired structural information.